Although a wealth of epidemiological studies indicate that high levels of HDL-C are associated with protection from cardiovascular disease, the recent failure of HDL-raising therapies at reducing clinical events has called the entire HDL paradigm into question. These studies underscore the critical need to better understand the mechanisms by which HDL exerts its atheroprotective effects, so that we can design therapeutics that harness these properties and provide maximum clinical benefit. Experimental studies in animals and humans indicate that therapies that increase the number of HDL particles or promote reverse cholesterol transport (RCT) confer atheroprotection. A major advance in our understanding of the regulation of HDL biogenesis and cholesterol efflux came from the identification by our lab and others those microRNA-33a/b (miR-33) represses key genes in these pathways, including ABCA1, ABCG1, NPC1. In preclinical studies in mice and non-human primates we showed that inhibition of miR-33 increases HDL-C, promotes RCT and regresses atherosclerotic plaques. These translational studies identify miR-33 pathway inhibition as a novel therapeutic strategy for targeting the HDL pathway, that is particularly promising as it increases both HDL biogenesis and reverse cholesterol transport, however the complete mechanisms by which anti-miR33 exerts its beneficial effects on HDL levels and functionality are not yet understood. There is thus a strong rationale to undertake a comprehensive analysis of miR-33 targeted pathways that generate an increased functional HDL particle to promote RCT and induce atherosclerosis regression. The aims proposed herein will identify new players in the miR-33 pathway that regulate HDL biogenesis/RCT and determine the target tissues responsible for the atheroprotective effects of anti-miR33. Together, these studies will enhance our understanding of the mechanisms of action of anti- miR-33 pathway inhibition, and provide important insight into the potential of this novel therapy for improving HDL functionality in treatment of cardiometabolic diseases.